Recent evidence indicates that both embryonic and adult stem cells have enormous therapeutic potential for cell therapy. In prior research, we have established that: a) therapeutically engineered stem cells migrate extensively to tumors and to infiltrating deposits in the brain and have apoptotic and antiangiogenic effects when transplanted into mouse models of glioma; and b) the dynamics of receptor targeted anti-tumor therapies and fate of stem cells can be visualized in real time in vivo. In this proposal, we will create toxin resistant human mesenchymal stem cells (MSC) for on-site delivery of targeted nanobodies and cytotoxic agents to simultaneously block proliferation and induce killing of tumor cells without affecting the normal brain. Specifically, MSC will be engineered to express targeted therapeutic proteins directed against overexpressed EGFR and specifically expressed IL13Ra2 and death receptors (DR)4/5 in glioma cells. In close collaboration with Henegouwen lab (Utrecht University, The Netherlands) which is leading efforts in developing low molecular weight, highly soluble EGFR specific nanobodies (EGFR-NB), we have recently shown that mammalian cells can be employed to express secretable bivalent EGFR nanobodies. We will initially express EGFR-NB in MSC and study the effect of EGFR-NB on EGFR signaling and cell proliferation in a panel of primary glioma cells and CD133+ primary brain tumor cells in an ongoing collaboration with Settleman lab (MGH, Boston). A number of studies have shown synergistic anti-tumor effects when EGFR signaling antagonists are combined with cytokines. Two different cytotoxic therapies based on selectively targeting glioma cells will be tested. In the first approach we will create toxin resistant MSC in an ongoing collaboration with Rich lab (B&W Hospital, Boston) and engineer MSC expressing EGFR-NB and IL13R-targeted Diphtheria toxin (DT) which is known to induce cell death by inhibition of protein synthesis through ADP-ribosylation of elongation factor-2 (EF-2). In the second approach we will create MSC expressing EGFR-NB and S-TRAIL, which we have extensively characterized and shown to selectively induce apoptosis via up-regulated death receptors (DR)-4/5 in proliferating glioma cells. Both approaches will be tested for their efficacy in culture and in vivo in established glioma lines. Based on these findings, we will utilize the most efficient therapeutic MSC in a highly invasive primary mouse model of glioma. We hypothesize that on site delivery of therapeutic MSC will result in simultaneous down-regulation of cell survival pathways and activation of death pathways thus resulting in enhanced eradication of gliomas. The integration of genetically engineered fluorescent and bioluminescent imaging markers and in vivo imaging in close collaboration with Weissleder lab (MGH, Boston) will allow us to follow delivery and fate of MSC and to asses their therapeutic efficacy in vivo These studies are expected to have a major impact in developing novel stem cell therapies that will eventually be compatible with clinical trials.